The lymphatic system is a specialized part of the circulation that is intimately involved in the maintenance of normal body fluid homeostasis. It is the primary means by which fluid and macromolecules are removed from the interstitium and returned to the venous circulation and it is thought to be one of the principal safety factors against gross edema formation. These tasks are accomplished through the involvement of valved vessels and active and passive lymph pumps. Fluid is actively pumped through the lymphatic system by the spontaneous generation of lymphatic contractions. The general objectives of the studies in this proposal will be to elucidate some of the mechanisms involved in the generation of spontaneous lymphatic contractions. The two key areas the proposed studies will focus on are: 1) the role of the endothelium and endothelial-smooth muscle interactions in spontaneous lymphatic contractions, and 2) the changes in intracellular calcium seen during spontaneous lymphatic contractions. These studies will be conducted in isolated, perfused collecting lymphatics (50-120 mu m in diameter) from the mesentery of the rat small intestine. The lymphatics will be dissected out of the tissue and cannulated. The pressure and flow through the vessel will be carefully controlled by varying the inlet and outlet pressure reservoirs. Lymphatic contractions will be monitored using videomicroscopic techniques. The lymphatic diameter will be continuously measured throughout the experiments. From the lymphatic diameter tracings, lymphatic contraction frequencies (F) and ejection fractions (EF) will be determined using a lymphatic cardiac cycle analogy. These two parameters (F and EF) are determining characteristics of the active lymph pump and will be used as functional indices of the spontaneous lymphatic contractions. The role of the endothelium and endothelial- smooth muscle interactions in affecting the spontaneous lymph pump will be investigated using techniques to inhibit and/or remove the lymphatic endothelium. Calcium is thought to play an important role in spontaneous lymphatic contractions. Intracellular levels of calcium in the lymphatics during spontaneous contraction will be monitored using the fluorescent calcium sensitive dye Indo-1. Using these techniques the following specific aims will be addressed: Determine the role of the endothelium and endothelial-smooth muscle cell interactions in: 1) the pressure sensitivity of the lymphatic pump; 2) the flow sensitivity of the lymphatic pump; 3) Measure the changes in intracellular calcium during spontaneous lymphatic contraction; 4) Determine the role of intra- and extracellular calcium stores in spontaneous lymphatic contraction; 5) Evaluate the role intracellular calcium plays in lymphatic contractile pacemaker activity. The results of these investigations should provide insight into some of the mechanisms involved in the generation of spontaneous lymphatic contractions and will be the basis for continued studies of the active lymph pump.